Pregnancy-related changes in the canine serum N-glycosylation pattern studied by Rapifluor HILIC-UPLC-FLR-MS

Canine reproduction differs from that of many other domestic animals, and increased knowledge on biochemical changes during canine pregnancy is important for investigations of infertility or subfertility. The total glycosylation pattern, i.e., the glycome, of body fluids reflects cellular status in health and disease. The aim of the present pilot study was to investigate pregnancy-related changes of the serum N-glycome in bitches. A method based on Rapifluor HILIC-UPLC-FLR-MS was optimized and applied for analysis and quantification of N-glycans in canine serum. Serum samples from six pregnant and five non-pregnant bitches, collected at four well-defined time points, were included. The levels of sialylated and galactosylated complex glycans were significantly elevated in serum from pregnant bitches, consistent with previous reports on human pregnancy. The levels of fucosylated and agalactosylated glycans decreased significantly in pregnant dogs. In non-pregnant dogs, the glycosylation pattern did not change during the cycle. Pregnancy is an inflammatory state, but our findings during canine pregnancy are quite the opposite to changes that have previously been described for dogs with a known parasitic infection. Evaluation of the canine glycome may thus be valuable in studies of canine pregnancy, possibly differing inflammatory changes related to pregnancy to those caused by an infection.


Glycan identification
A method based on RapiFluor-labelling and subsequent HILIC-UPLC-FLR-MS analysis 22 was developed and optimized for N-glycans in canine serum.A representative chromatogram is shown in Fig. 1.Structural assignment of glycan peaks was performed by mass spectrometry, tandem mass spectrometry and glycan reference standard analysis.In total, glycan structures were assigned to 50 peaks (Fig. 1, Supplementary Fig. S1, Supplementary Table S1-S4).The identified glycans were mainly complex diantennary structures and the two most abundant N-glycan species were A2G2S2 and FA2.The structural assignment and elution pattern were compared to and demonstrated to be in line with previously reported N-glycan analysis of dog serum 21 .
The glycans were categorized into different groups to allow for further evaluation.The selected groups were sialylated, fucosylated, high mannose, terminally galactosylated, galactosylated and agalactosylated complex glycans, and the categorisation of each glycan is described in Supplementary Table S1.Galactosylated glycans constituted around 75% of the total N-glycan content, and around 70% of the N-glycans were sialylated (refer to Table 1).Approximately 30% of the N-glycans were found to be fucosylated.The majority of the fucosylated glycans were confirmed to be core-fucosylated by MS/MS analysis and detection of the diagnostic ion at m/z = 679.7,analogous to the core fucose diagnostic peak previously reported for procainamide labeling 23 .Furthermore, 5% were high mannose structures, 18% were agalactosylated complex glycans and 13% were terminally galactosylated.

Pregnancy-related changes
The serum N-glycome of eleven bitches was determined at three individual timepoints during the oestrous cycle, i.e., Day 1, Day 29 and Day 43.An extra timepoint, Day 22, was included for four pregnant and four non-pregnant dogs.The dominating glycan was for all dogs and all timepoints A2G2S2, constituting on average 49% of the total N-glycan content at Day 1 both for pregnant and control bitches.The second most abundant glycan was FA2, constituting on average 13% and 15%, respectively for the two groups.The abundances of these two glycans at the four timepoints for pregnant and control bitches are shown in Fig. 2. While the levels of both glycans remained stable over time for control bitches, the relative abundance of A2G2S2 increased by approximately 6 percentage points and the levels of FA2 decreased by 3 percentage points for pregnant bitches.The levels of A2G2S2 and FA2 for all individual dogs at the four timepoints are shown in Fig. 3, and the corresponding data is available in Supplementary Tables S5-S6.It is noted that the changes determined to be statistically significant (Fig. 2) were consistent for all individual pregnant dogs (Fig. 3).For A2G2S2, there was an increase as compared to Day 1 for all dogs and timepoints, except for one dog Day 43 (Fig. 3A).For FA2, a decrease as compared to Day 1 was detected for all dogs and timepoints, except for one dog Day 22 (Fig. 3C).No common trends were noted for control dogs (Fig. 3B and D).Except for A2G2S2 and FA2, no other individual glycan constituted more than 3% of the total N-glycan content at Day 1 on average for all dogs.These glycans were therefore not evaluated individually, but in groups of related glycans.Changes over time were evaluated for sialylated, fucosylated, high mannose, terminally galactosylated, galactosylated and agalactosylated complex glycans.The results are summarised in Table 1 and numerical values for all dogs are included in Supplementary Tables S7-S12.Significant changes were confirmed for sialylated, fucosylated, galactosylated and agalactosylated complex N-glycan, applying repeated measures ANOVA and post-hoc analysis by Dunnett's correction for multiple comparison.In fact, significant changes were Table 1.Changes of serum N-glycosylation in pregnant and control dogs.The differences in percentage points compared to the optimal day of mating (Day 1) are monitored for Days 22, 29 and 43 during pregnancy and the corresponding days for the control dogs.Significant changes (p < 0.05) were confirmed for sialylated, fucosylated, galactosylated and agalactosylated complex N-glycan, applying repeated measures ANOVA and post-hoc analysis by Dunnett's correction for multiple comparison.NS denotes statistically non-significant changes.www.nature.com/scientificreports/observed both at Day 29 and Day 43 for all four glycan groups, increased abundance was noted for sialylated and galactosylated glycans, while there was a decrease for fucosylated and agalactosylated complex glycans.The alterations were also observed for these glycan groups at Day 22, even though the changes were solely found statistically significant (p < 0.05) for the fucosylated glycans at this time point.At Day 29, increased abundance of sialylated and galactosylated glycans and decreased levels of fucosylated and agalactosylated complex glycans were confirmed for all six pregnant dogs, as shown by the min-max range.No significant changes in the levels of any of the glycan groups were noted for control bitches at the corresponding days in the oestrous cycle.Supplementary Table S13 shows the HILIC-FLR chromatograms for the Day 1 and Day 29 serum samples for all 11 dogs, and the abundances of all identified N-glycans are tabulated.

Method performance and optimization
The RapiFluor-HILIC-UPLC-FLR-MS method was optimized with respect to sample preparation and chromatography.A target volume equivalent to 0.75 µL serum was selected and the robustness of the method was studied by varying the serum volume.Additionally, during previous glycan analysis in our laboratory the importance of buffer exchange prior to N-glycan deglycosylation and labelling was noted 24 , whereby this aspect was additionally studied.It was concluded that the method provided consistent results over the studied range, that the selected target volume was appropriate, and that filtration of samples was not needed (Supplementary Fig. S2).
The chromatographic separation was optimized by increasing the length of the gradient and by increasing the concentration of ammonium formate in the mobile phase.The latter gave a significant improvement of the peak shape of late eluting, sialylated glycans, presumably by mitigating detrimental ionic secondary interactions (Supplementary Fig. S3).A higher ammonium formate concentration could however lead to decreased MS signals, due to ion suppression, but the improved chromatography significantly counteracted any such effects, with a two-fold increase of peak height for A2G2S2 (200 mM vs 50 mM ammonium formate, Supplementary Fig. S4).
The variability of the optimized method was evaluated by repeated sample preparation and analysis of an assay control sample, i.e., a serum sample from a non-pregnant bitch.The sample was prepared at 10 different occasions using four individual RapiFluor-MS N-Glycan Kits, referred to as Kit 1-4.The resulting intermediate precision data are presented in Table 2.A higher precision was obtained for samples prepared within a kit, compared to the overall precision attained from all four kits.Additionally, repeatability precision was obtained by repeated injection of the assay control sample (Supplementary Table S14).From this data it was concluded that sample preparation introduced most of the variability of the obtained results, but that the overall precision still was considered acceptably low.

Discussion
We here describe, for the first time, pregnancy-related changes in the total serum glycosylation pattern for the dog.The levels of sialylated glycans were elevated during canine pregnancy, which is in line with what has been confirmed for human pregnancy 19,25 .Few studies have been conducted on other species, but increased sialylation has also been reported for the camel 26 .The increase for dogs was observed already at Day 22 and a significant increase of around 5 percentage points was detected on Day 29, remaining during Day 43.In addition, the total proportion of galactosylated glycans was elevated Day 29, similar to the situation in human pregnancy, in which the levels of IgG galactosylation have been demonstrated to increase 27,28 .In contrast, the levels of fucosylated and agalactosylated glycans decreased Day 29.This is similar to the situation in the camel, in which a decrease of fucosylated glycans has been described 26 .It should be noted that pregnancy-associated changes are not straightforward to compare between species since the overall glycan pattern differs between mammals 21,29 and since the pregnancy length and placenta types are different.However, the findings of the present study further support that at least some glycosylation events are similar between species.
While the increase in sialylation and galactosylation during pregnancy generally agrees with what has been observed for other species, the results are opposite to what has been reported for dogs infected with Dirofilaria immitis 20 .Changes in the overall N-glycan pattern can be caused both by changes in the relative abundances of Table 2. Intermediate precision data obtained from sample preparation and analysis of the assay control sample, performed on 10 different occasions, using 4 different sample preparation kits (data from kit 4, n = 1, not separately displayed)..
In addition, an increase in glycoprotein acetyls (GlycA), as measured by NMR, has been observed during canine mid-pregnancy 31,32 .The levels of GlycA can be regarded as a combined response for several glycoprotein APPs, and GlycA has been suggested an inflammatory biomarker for systemic inflammation 33 .It is thus well-established that changes related to abundant serum glycoproteins occur in mid-pregnancy.However, our study is the first to monitor the changes at the level of individual glycans and glycan groups.In dogs infected by Dirofilaria immitis, the decrease in sialylation was considered most likely related to changes in the overall glycoprotein pattern, in particular an increase in the IgG level, resulting in higher relative abundance of FA2 but lower abundance of A2G2S2 20 .The changes in N-glycan pattern observed in our study could be due to changes in other glycoprotein levels, as compared to the dogs infected by Dirofilaria immitis, but it is also likely that they are related to changes in glycan microheterogeneity of abundant serum proteins, including APPs.The method chosen for N-glycomics analysis in the present study, RapiFluor-HILIC-UPLC-FLR-MS, was initially developed for analysis of individual, purified glycoproteins 22 , but high throughput applications on human plasma 34,35 have demonstrated good repeatability and robustness in more complex samples.To the best of our knowledge, this is the first application of RapiFluor-labeling of glycoproteins in canine serum.The sample consumption of the method developed in our laboratory is very low, around one µL of serum is sufficient for the analysis.This volume is minor as compared to the volume of blood samples collected from dogs; typically measured in mL.Thus, glycan analysis can be performed on serum samples extracted for other diagnostic purposes, which is in line with the 3Rs: Replace, Reduce, Refine.In addition, results from another study indicate that the N-glycan profile of canine serum remains stable during long-term storage at -80 °C for at least 25 years 20 .Thus, investigations of changes in the glycosylation pattern would be possible also for rare conditions where few samples can be collected over time.In the current study, samples were collected over a rather short total time period of 1.5 years and for each individual dog, blood samples were obtained during 42 days.
The method variability was studied by repeated sample preparation of an assay control, followed by HILIC-UPLC-FLR-MS analysis (Table 2).The variability within a sample preparation kit was lower than the total variability, encompassing four different kits.To avoid unwanted variability due to sample preparation, samples from individual dogs were therefore always prepared within a kit.The repeatability of the LC-method was of such a degree that highly robust retention times were obtained which facilitated identification of glycans in serum samples when using glycan reference standards (Supplementary Table S14).Additionally, the repeatability precision of the LC-method with respect to glycan abundance was high enough to enable single injection of samples, thereby avoiding time consuming replicate injections (Supplementary Table S14).
For healthy humans, the serum N-glycan profile has been demonstrated to be remarkably stable for individuals, but relatively variable within the population 17 .Our results confirm that the serum N-glycome is stable for non-pregnant bitches during the oestrous cycle.No significant changes were observed for any of the N-glycan groups, and the average difference in abundance as compared to Day 1 were for most glycan groups less than one percentage point (Table 1).Analogous to the situation in humans, the variability between dogs was demonstrated to be larger and the repeated measures design of the study was therefore considered to be advantageous.The significant changes observed for pregnant bitches were 5 percentage points for sialylated and fucosylated glycans and around 4% for galactosylated and agalactosylated complex glycans.Due to the variability observed between dogs it is suggested that if the N-glycan pattern, in future, will be used for diagnostic purposes, serum samples from the bitches should also be collected before mating to establish reference levels.It should be noted that the trends established to be statistically significant were confirmed for all six individual dogs at Day 29, i.e., there was an increase in the abundance of sialylated and galactosylated glycans and a decrease in fucosylated and agalactosylated glycans, even though the magnitude of the changes differed between dogs.
From a diagnostic point of view, the results obtained on N-glycosylation profile in this study are interesting.The changes observed in the glycan pattern, including altered levels of the individual glycans A2G2S2 and FA2, increased sialylation and galactosylation and decreased fucosylation, occurred already at D22, at the same time as we have previously described changes in the APPs fibrinogen and CRP 8,9 .However, the glycan pattern for pregnant dogs differs from that previously described for dogs with a known infection with D. immitis 20 , although infection with D. immitis has been described to cause an acute phase response with associated changes in the APPs 36 .This suggests that although changes in the glycan pattern were detected at the corresponding times to previously described changes in APP concentrations, the glycan pattern may not be caused by inflammatory changes or that there is a change in microheterogeneity of the APPs that differs between pregnancy and other inflammatory states.From a diagnostic point of view this is beneficial, as this means that the glycan pattern possibly is a more specific marker for pregnancy than the concentration of APPs, as the latter will change because of any inflammatory cause.However, the changes in glycan pattern related to bacterial or viral infections should also be investigated.The findings of this exploratory study should also be confirmed in an extended study including more dogs.

Statistical analysis
Statistical analysis was performed in Minitab©.The glycan/glycan group levels were evaluated using repeated measures ANOVA for pregnant (n = 6) and non-pregnant (n = 5) dogs, respectively, at four timepoints; Day 1, Day 22, Day 29 and Day 43.Post-hoc analysis was performed applying the Dunnett's correction for multiple comparison, setting Day 1 as the reference day.P-values < 0.05 were considered statistically significant.

Ethical considerations
The study was approved by the Uppsala Ethical Committee of Animal Experimentation (C23/9) and the Swedish Board of Agriculture (31-1365/09).All experiments were performed in accordance with relevant guidelines and regulations.The dog owners gave their informed written consent.The study was performed according to these approvals and to the ARRIVE guidelines.The methodology described and the conclusions drawn in this paper are the authors' opinion and not necessarily the standpoint of the Swedish Medical Products Agency.

Fig. 2 .Fig. 3 .
Fig. 2. Abundance of the two dominating N-glycans in canine serum during pregnancy shown as % of the total N-glycan content.Box plots for control (C, blue boxes, n = 5) and pregnant bitches (P, red boxes, n = 6) sampled at the same days of the oestrous cycle are shown for glycan A2G2S2 (A) and glycan FA2 (B).Asterisks (*) indicate significant changes as compared to Day 1.For pregnant bitches, an increase of 6 percentage points was observed for A2G2S2 (p < 0.05 Day 29).A significant decrease of 3 percentage points was detected for FA2 for all three time points (p < 0.05).No changes over time were observed for control bitches. https://doi.org/10.1038/s41598-024-71352-zwww.nature.com/scientificreports/